Project Summary Understanding how microbes adapt to a novel environment is essential to our understanding of how microbes respond during acute infection or long-term disease. Specifically, the genetic architecture underlying the constraints on producing and maintaining molecular variation within bacterial genomes directly influences the population's potential for adaptation and eventual evolutionary fate. Upon colonization, competition for resources within populations drives adaptation, which can result in cooperation, innovation, or interference among individuals within the population. Previous studies have shown that microbial subpopulation structure can be established as a potential adaptive outcome in heterogeneous environments, but have yet to investigate the long-term stability of this structure and the genetic framework that allows subpopulation structure to be maintained. To bridge this gap of knowledge I propose to: (i) conduct genetic and environmental manipulations of Escherichia coli K-12 MG1655 in order to determine the importance of biofilm formation for establishment of subpopulation structure, (ii) isolate individual clones from E. coli populations experiencing different degrees of starvation to identify how nutrient limitation affects the stability of subpopulation structure, and (iii) analyze spent media from these clones to define the different nutrient utilization strategies within and among populations. In addition to the populations created for the specific purpose of this study, sampling will also occur from 320 long-term E. coli populations which will have been maintained up to 6 years and over 17,000 generations at the conclusion of this fellowship, generating a well of opportunity for future research questions through which I aspire to establish a career as an independent investigator.